Mass spectrometry has been widely used for sequencing and quantitation of proteins and peptides. To identify proteins, for instance, peptides produced by enzyme digestion are ionized by either Matrix-Assisted Laser Desorption/Ionization(MALDI) or Electrospray Ionization(ESI), and then their masses can be measured by means of a mass spectrometer to characterize the protein. More exactly, some peptides are further cleaved into fragments to identify the peptide sequence.
For the quantification of proteins and peptides by mass spectrometry, a number of stable isotope tags have been chemically introduced as markers into proteins or peptides of interest. Chemical tags differentially labeled with isotopes are incorporated into the same samples to be analyzed, and the mass of each sample can be distinguished due to the mass difference of the isotopes in the resulting mass spectra or tandem mass spectra, thus allowing protein quantification by the comparison of their relative intensities.
Recently, the isobaric chemical tagging strategy has been used for simultaneous protein quantitation and sequencing. In US 2005/0148087 and WO 2005/068446, disclosed are isobaric reagents labeled with isotopes, which bind with peptide to display quantitation signals in tandem mass spectrometry.
However, the labeling reagents used in the known methods are problematic in that expensive carbon, nitrogen and oxygen isotopes are used, thus carrying high cost. In addition, another drawback is that because of the limited signal mass window, unexpected chemical noise may hinder the analysis. Therefore, there is a need for novel isobaric labeling reagents incorporating low-cost hydrogen isotopes for simultaneous peptide sequencing and protein quantitation. Further, there is a need for novel isobaric variable mass labeling reagents that provide tunability not only in mass window of quantitation signals but also in property of peptides, thus applicable to a wide range of biomolecules.
The present inventors have suggested a novel isobaric labeling reagent based on dipeptide, mass-balanced 1H/2H-isotope tag (MBIT) which only employs hydrogen isotopes and offers tunability in quantitation signal mass window, disclosed in Korean Patent Application No. 2008-0070272. Further, they have demonstrated that the replacement of the mass-tunable group of the 2-plex isobaric labeling reagent with other natural amino acid side chains having various properties offers possibilities of tuning the signal mass window and its property, disclosed in Korean Patent Application No. 2009-0019444. Various MBITs having different amino acid side chains showed up to ten-fold difference in the quantitation signal intensities due to dissimilar chemical properties of the amino acid side chains. To achieve better performance of the MBIT reagents in simultaneous multiplexed quantitation, it is necessary to use the MBIT reagents having similar chemical properties but different quantitation signals in a combination of two or more thereof. Accordingly, for simultaneous multiplexed protein quantification, a variety of MBIT reagents having identical property is needed to provide similar quantitation signal intensity. Thus, they have suggested mass- and property-tuned variable mass isobaric labeling reagents, a set of the labeling reagents, and analysis methods for simultaneous quantitation, disclosed in Korean Patent Application No. 10-2009-0054540.